SUMMARY. Instruction of T cells to be tolerant to self occurs in the thymus through interactions of thymocytes with the epithelial cell compartment. While the thymus serves as the primary site of immunologic T cell tolerance to self-antigens and production of thymic T regulatory (tTreg) cells, microbial communities in the gut also regulate the function of T effector (Teff) cells and production of peripheral Treg cells (pTreg). Microbial colonization of mucosal tissues in early life facilitates tolerance to commensal and environmental antigens. Abnormal colonization during this period can have long-term consequences contributing to development of mucosal and systemic disease later in life. The mechanisms that regulate perinatal immune- system-microbiota interactions to achieve long-term homeostasis are poorly defined. We previously generated mice (Traf6?TEC) whose thymus was devoid of medullary thymic epithelial cell (mTECs). mTEC depletion had a two-faceted effect on the T cell output: generation of autoreactive T cells and a 50% reduction in the numbers of tTregs. The reduction in tTregs in Traf6?TEC mice in turn associated with: 1) reduced numbers of Foxp3+ Tregs and T follicular (Tf) cells in the Peyer?s Patches (PP) of the small intestine and small intestinal inflammation; 2) skewed production of IgA-coated bacteria; and 3) altered microbial composition in the gut of knockout animals. Together, these results suggest that the aberrant T cell compartment generated in the thymus of Traf6?TEC mice and associated changes in the microbiota may adversely impact survival of newborn Traf6?TEC mice. In this proposal we will use the Traf6?TEC mouse model to better understand how T cells and the gut microbiota influence each other to establish perinatal organ-specific tolerance. We will test the following hypothesis: Impaired tTreg cell selection in Traf6?TEC mice, induces changes in their gut microbiota that cannot support induction of perinatal tolerance and viable progeny. Perinatal exposure of knockout pups to normal microbiota is required for tolerance induction and survival. To test this hypothesis we will: 1) examine whether exposure to normal microbiota is sufficient for promoting survival of newborn Traf6?TEC mice and identify microbial communities from WT mice that rescue Traf6?TEC neonate survival; 2) examine whether induction of neonatal tolerance is compromised in newborn Traf6?TEC mice that are not exposed to normal microbiota; and 3) examine if the altered T cell compartment of Traf6?TEC mice acts as a genetic determinant of intestinal dysbiosis and neonatal lethality. Defining early-life tolerance mechanisms could have a profound impact on our understanding of human autoimmune disease development and may help us devise novel strategies for managing and/or treating T cell mediated autoimmune diseases.